Methods for the preparation of liposomes

ABSTRACT

Provided herein are methods for preparing liposomes and uses thereof. In certain embodiments, liposomes are prepared without using heat, organic solvents, proteins, and/or inorganic salts in the process. In certain embodiments, the liposomal preparation contains one or more active agents. In certain embodiments, the liposomal preparations are used in the treatment of diseases or disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119 of U.S.Provisional Patent Appl. No. 61/245,185, filed Sep. 23, 2009, thedisclosure of which is incorporated by reference in its entirety herein.

BACKGROUND

The bioavailability of a pharmaceutical drug depends largely in part onthe solubility and stability of the drug. Many methods have beenemployed to improve bioavailability of a drug, including, but notlimited to, pH adjustment, associating the drug in micelles ofdetergents, solubilization in an organic solvent, complexation withcyclodextrin or other polymers, and encapsulating the drug in a liposomebilayer (Strickley, R. G., Pharmaceutical Research, No. 21, 2004:201-230). Either the drug itself or the excipients used to solubilizethe drug may have side effects such as allergic reaction or hemolysis.

It is known that the solvents (e.g., ethanol, propylene glycol,polyethylene glycol, dimethylacetamide, dimethylsulfoxide (“DMSO”)),complexing agents (for example, nicotinamide), and surfactants (forexample, sodium oleate) are hemolytic and are therefore undesirable foruse in injectable solutions. Other limitations to using organic solventsin injectable products include precipitation, pain, and inflammationupon injection.

Liposomes are microscopic lipid vesicles that are composed of a centralaqueous cavity surrounded by a lipid membrane formed by concentricbilayer(s) (lamellas). Liposomes are able to incorporate hydrophilicsubstances (in the aqueous interior) or hydrophobic substances (in thelipid membrane). Liposomes can be unilamellar vesicles (“UMV”), having asingle lipid bilayer, or multilamellar vesicles (“MLV”), having a seriesof lipid bilayers (also referred to as “oligolamellar vesicles”). Themultilamellar vesicles typically range in size from 0.2 μm to 10 μm indiameter. See e.g., WO 98/006882. Although anti-hemolytic measures arecommonly taken in formulations, maintaining a sufficient amount ofliposome in formulation may not be feasible due to the incompatibilityof the liposome with an excipient, or the instability of the liposome inthe formulation. Further, reconstituting lyophilized formulationscontaining hydrophobic drugs is often difficult. Moreover, liposomes arenot stable in formulations containing concentrated organic solvents.

Unilamellar vesicles with a diameter of less than 0.2 μm (e.g. between0.02 and 0.2 μm) are commonly known as small unilamellar vesicles(“SUV”). Unilamellar vesicles with a diameter greater than 0.45 μm (insome cases greater than 1 μm) are commonly known as large unilamellarvesicles (“LUV”).

The bilayer(s) of liposomes most often comprise phospholipids, but mayalso comprise lipids including but not limited to fatty acids, fattyacid salts and/or fatty alcohols. The properties of the liposomesdepend, among other factors, on the nature of the constituents.Consequently, if liposomes with certain characteristics are to beobtained, the charge of its polar group and/or the length and the degreeof saturation of its fatty acid chains must be taken into account.

In addition, the properties of liposomes may be modified, e.g., toincorporate cholesterol and other lipids into the membrane, change thenumber of lipidic bilayers, or covalently join natural molecules (e.g.,proteins, polysaccharides, glycolipids, antibodies, enzymes) orsynthetic molecules (e.g., polyethyl glycol) to the surface. There arenumerous combinations of phospholipids, optionally with other lipids orcholesterol, in an aqueous medium to obtain liposomes. Depending on themethod of preparation and the lipids used, it is possible to obtainvesicles of different sizes, structures, and properties.

Another important parameter to consider with respect to the formation ofliposomes is the rigidity of the lipidic bilayer. The hydrated lipidthat focus part of the bilayer may be in either a liquid-crystalline(fluid) or gel state. As the temperature increases, the gel state isconverted into the liquid-crystalline state. This occurs at atemperature known as the transition temperature (Tc), which is specificto each lipid. The Tc is directly proportional to chain length andinversely proportional to the degree of unsaturation of the fatty acidsand depends on the nature of the polar group.

Despite this, common methods in the preparation of lipid vesicles, suchas liposomes, comprise evaporating an organic solvent in which thelipids are dissolved and then dispersed in an optionally bufferedaqueous solution. One exemplary method, known as the Bangham method, wasoriginally described in Bangham et al., J. Mol. Biol., 11:238-252(1965). Variations of the Bangham method are known by those skilled inthe art, some of which are described below.

Hydration of a thin lipidic layer. Starting with the organic solution ofthe constituent lipids of the bilayer, a lipidic film is preparedthrough removal of organic solvent, which can be achieved by means ofevaporation (e.g., at reduced pressure in a rotary evaporator) or bylyophilization. The dry lipidic film obtained is hydrated by adding anaqueous solution and agitating the mixture at temperatures above the Tc.

Reverse-phase evaporation. Starting with the organic solution of theconstituent lipids of the bilayer, a lipidic film is prepared throughremoval of the organic solvent. The system is purged with nitrogen andthe lipids are re-dissolved in a second organic solution, usuallyconstituted by diethyl ether and/or isopropyl ether. The aqueous phaseis added to the re-dissolved lipids. The system is maintained undercontinuous nitrogen. A gel is formed by removing the second organicsolvent.

Solvent injection. The lipids, dissolved in an organic solvent, areinjected slowly into an aqueous solution. The organic solvent used isoften a water-miscible solvent, and the aqueous solution may be warmed.

Additional methods for the preparation of multilamellar vesicles can befound, e.g., in Szoka and Papandjopoulos, Ann. Rev. Biophys. Bioeng., 2:467-508 (1980), and Dousset and Douste-Blazy, Les Liposomes, Puisieuxand Delattre, Editors, Tecniques et Documentation Lavoisier, Paris, pp.1-73 (1985).

Further, when the incorporation of more than one lipid is desired, thelipids should remain homogeneously distributed in the liposomalvesicles. Traditionally, this is achieved by previously dissolving thelipids in an organic solvent and using the resulting organic solvent forpreparing the liposomes.

U.S. Pat. No. 4,508,703 describes a method for obtaining powderymixtures of at least one amphyphilic lipid and, optionally, at least onecomponent of a hydrophobic or partially hydrophobic nature, a methodwhich includes dissolving the components of the mixture in at least oneorganic solvent and atomizing the obtained solution into an inert gas.The method permits the preparation of lipidic mixtures which can beeasily dispersed in an aqueous medium but does not avoid the use oforganic solvents.

WO 92/10166 describes a method for preparing liposomes with an elevatedencapsulation capacity. The method permits the use of mixtures oflipids; however, the mixture is obtained by means of previousdissolution of the lipids in an organic solvent and subsequentevaporation. In addition, the contact between the lipids and the aqueoussolution of active agent is carried out at a temperature above the Tc.

Moreover, it is reported that, where liposomes are made without usingorganic solvents, other manipulations, which may result in formulationswith certain unfavorable characteristics, are generally required. Forexample, U.S. Pat. App. Pub. No. 2008/0274172 describes methods ofpreparing liposomes containing at least two phospholipids without usingorganic solvents. However temperatures above the Tc were used to obtainstable liposomes from aqueous solutions containing inorganic salts.

Consequently, existing methods for preparing liposomes utilize organicsolvents, protein, inorganic salts, and/or heat. Due to their toxicityand flammability, organic solvents are undesirable in the preparation ofliposomes for pharmaceutical, cosmetic and other uses. Moreover, the useof organic solvents and proteins has negative repercussions in terms ofproduction costs, safety, work hygiene and the environment. Similarly,the use of heat in the preparation of liposomes is undesirable in termsof production costs, safety, and the environment. The use of inorganicsalts in the preparation of liposomes is undesirable as the introductionof inorganic salts increases the size of the liposome and/or results ina more turbid formulation. See e.g. Castile et al., InternationalJournal of Pharmaceutics, 1999, vol. 188, issue 1, pp. 87-95. Thus,there is a need for a method for preparing liposomes without the use ofundesirable agents and procedures.

SUMMARY

Provided herein are methods for preparing liposomes. In certainembodiments, liposomes are prepared without using heat, organicsolvents, proteins, and/or inorganic salts in the process.

In one embodiment, a method is provided for the preparation ofliposomes, the method comprising:

(a) Combining one or more lipids in an aqueous medium at ambienttemperature;

(b) Dispersing the lipids in the aqueous medium; and

(c) Adding one or more sugars to the resulting mixture, thereby forminga solution of liposomes.

In certain embodiments, the liposomal preparation contains one or moreactive agents. In certain embodiments, the active agent is apharmaceutical agent. In certain embodiments, active agents include, butare not limited to lapachone; transferrin; cyclosporine; colchicine; andcombinations of lapachone and transferrin in a single liposomeformulation.

In certain embodiments, the one or more active agents are added afterthe liposome or solution of liposomes has been prepared. In otherembodiments, the one or more active agents are added during formation ofthe liposome.

Also provided herein are uses of liposomal preparations in the treatmentof diseases or disorders. In one embodiment, the liposomal preparationis suitable for parenteral administration to a patient suffering fromsaid disease or disorder. In one embodiment, the patient is a human.

DEFINITIONS

As used herein, and unless otherwise specified, “lipid” is understood tobe a fatty acid, fatty acid salt, fatty alcohol, or phospholipid. Lipidsmay also be read to include sterols, including, but not limited to,cholesterol; sphingolipids, including, but not limited to,sphingomyelin; glycosphingolipids including, but not limited to,gangliosides, globocides and cerebrosides; and surfactant aminesincluding, but not limited to, stearyl, oleyl and linoleyl amines.

As used herein, and unless otherwise specified, “phospholipid” isunderstood to be an amphyphilic derivative of glycerol, in which one ofits hydroxyl groups is esterified with phosphoric acid and the other twohydroxyl groups are esterified with long-chain fatty acids that can beequal to or different from each other and can be saturated orunsaturated. A neutral phospholipid is generally one in which the otherphosphoric acid hydroxyl is esterified by an alcohol substituted by apolar group (usually hydroxyl or amino) and whose net charge is zero. Aphospholipid with a charge is generally one in which the otherphosphoric acid hydroxyl is esterified by an alcohol substituted by apolar group and whose net charge is positive or negative.

Examples of phospholipids include, but are not limited to phosphatidicacid (“PA”), phosphatidylcholine (“PC”), phosphatidylglycerol (“PG”),phophatidylethanolamine phophatidylinositol (“PI”), andphosphatidylserine (“PS”), sphingomyelin (including brainsphingomyelin), lecithin, lysolecithin, lysophosphatidylethanolamine,cerebrosides, diarachidoylphosphatidylcholine (“DAPC”),didecanoyl-L-alpha-phosphatidylcholine (“DDPC”),dielaidoylphosphatidylcholine (“DEPC”), dilauroylphosphatidylcholine(“DLPC”), dilinoleoylphosphatidylcholine, dimyristoylphosphatidylcholine(“DMPC”), dioleoylphosphatidylcholine (“DOPC”),dipalmitoylphosphatidylcholine (“DPPC”), distearoylphosphatidylcholine(“DSPC”), 1-palmitoyl-2-oleoyl-phosphatidylcholine (“POPC”),diarachidoylphosphatidylglycerol (“DAPG”),didecanoyl-L-alpha-phosphatidylglycerol (“DDPG”),dielaidoylphosphatidylglycerol (“DEPG”), dilauroylphosphatidylglycerol(“DLPG”), dilinoleoylphosphatidylglycerol,dimyristoylphosphatidylglycerol (“DMPG”), dioleoylphosphatidylglycerol(“DOPG”), dipalmitoylphosphatidylglycerol (“DPPG”),distearoylphosphatidylglycerol (“DSPG”),1-palmitoyl-2-oleoyl-phosphatidylglycerol (“POPG”),diarachidoylphosphatidylethanolamine (“DAPE”),didecanoyl-L-alpha-phosphatidylethanolamine (“DDPE”),dielaidoylphosphatidylethanolamine (“DEPE”),dilauroylphosphatidylethanolamine (“DLPE”),dilinoleoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine(“DMPE”), dioleoylphosphatidylethanolamine (“DOPE”),dipalmitoylphosphatidylethanolamine (“DPPE”),distearoylphosphatidylethanolamine (“DSPE”),1-palmitoyl-2-oleoyl-phosphatidylethanolamine (“POPE”),diarachidoylphosphatidylinositol (“DAPI”),didecanoyl-L-alpha-phosphatidylinositol (“DDPI”),dielaidoylphosphatidylinositol (“DEPI”), dilauroylphosphatidylinositol(“DLPI”), dilinoleoylphosphatidylinositol,dimnyristoylphosphatidylinositol (“DMPI”) dioleoylphosphatidylinositol(“DOPI”), dipalmitoylphosphatidylinositol (“DPPI”),distearoylphosphatidylinositol (“DSPI”),1-palmitoyl-2-olcoyl-phosphatidylinositol (“POPI”),diarachidoylphosphatidylserine (“DAPS”),didecanoyl-L-alpha-phosphatidylserine (“DDPS”),dielaidoylphosphatidylserine (“DEPS”), dilauroylphosphatidylserine(“DLPS”), dilinoleoylphosphatidylserine, dimyristoylphosphatidylserine(“DMPS”), dioleoylphosphatidylserine (“DOPS”),dipalmitoylphosphatidylserine (“DPPS”), distearoylphosphatidylserine(“DSP”), 1-palmitoyl-2-olcoyl-phosphatidylserine (“POPS”), diarachidoylsphingomyelin, didecanoyl sphingomyelin, dielaidoyl sphingomyelin,dilauroyl sphingomyelin, dilinoleoyl sphingomyelin, dimyristoylsphingomyelin, sphingomyelin, dioleoyl sphingomyelin, dipalmitoylsphingomyelin, distearoyl sphingomyelin, and1-palmitoyl-2-oleoyl-sphingomyelin.

As used herein, and unless otherwise specified, “encapsulate” or“encapsulation” is understood to be the process of incorporating anactive agent into liposomes or liposomal vesicles. The encapsulatedactive agent can remain in the aqueous interior or associate withmembranes.

As used herein, and unless otherwise specified, the term “enhance” or“enhancing,” when used in connection with the solubility of a compound,means that the methods provided herein result in the increasedsolubility of the compound as compared to the solubility of the samecompound in water. Specifically, the term “enhance” or “enhancing” meansthat, when the methods provided herein are used, the solubility of acompound increases about 20 percent or more, about 40 percent or more,about 60 percent or more, about 80 percent or more, about 100 percent ormore, or about 200 percent or more of the solubility of the samecompound in a reference solvent. In some embodiments, the referencesolvent is water.

As used herein, and unless otherwise specified, the term “hydrophobiccompound” means a compound with little or no water solubility. In someembodiments, a hydrophobic compound has an intrinsic water solubility(i.e., water solubility of the unionized form) of less than about 20percent by weight, about 15 percent by weight, about 10 percent byweight, about 5 percent by weight, about 1 percent by weight, about 0.1percent by weight or about 0.01 percent by weight. In other embodiments,a hydrophobic compound has an intrinsic water solubility of less thanabout 10 mg/mL, about 7 mg/mL, about 5 mg/mL, about 3 mg/mL, about 1mg/mL or about 0.1 mg/mL.

As used herein, or unless otherwise specified, the terms “aqueousmedium” or “aqueous media” include any water based medium, e.g., water,saline solution, a sugar solution, a transfusion solution, a buffer, andany other readily available water-based medium. Further, an aqueousmedium may contain one or more water soluble organic solvents. In thecase of a parenteral solution, an aqueous medium is preferably sterileand suitable for use as a carrier of an active agent. Examples ofaqueous media include, but are not limited to, water for injection,saline solution, Ringer's solution, D5W, or other solutions ofwater-miscible substances such as dextrose and other electrolytes.

As used herein, and unless otherwise specified, the term “fatty acid”means a compound whose structure is a carboxylic group attached to ahydrocarbon chain having one or more carbon atoms. The hydrocarbon chainmay be saturated or unsaturated (i.e., alkyl, alkenyl or alkynylhydrocarbon chains). Also, the hydrocarbon chain may be straight orbranched. Moreover, in some embodiments, hydrogens in the hydrocarbonchain may be substituted.

As used herein, and unless otherwise specified, the term “fatty alcohol”means a compound whose structure is an alcohol group attached to ahydrocarbon chain having one or more carbon atoms. The hydrocarbon chainmay be saturated or unsaturated (i.e., alkyl, alkenyl or alkynylhydrocarbon chains). Also, the hydrocarbon chain may be straight orbranched. Moreover, in some embodiments, hydrogens in the hydrocarbonchain may be substituted.

As used herein, and unless otherwise specified, the term “fatty acidsalt” means a compound formed from a reaction between a fatty acid andan inorganic/organic base. In addition, the term encompasses a compoundformed from a reaction between a fatty alcohol and an inorganic/organicacid. Examples of such acids include, but are not limited to, sulfuricand phosphoric acid. The hydrocarbon chain of the fatty acid salt may besaturated or unsaturated (i.e., alkyl, alkenyl or alkynyl hydrocarbonchains). In addition, the hydrocarbon chain may be straight or branched.Moreover, in some embodiments, hydrogens in the hydrocarbon chain may besubstituted.

As used herein, and unless otherwise specified, the term “substituted”means a group substituted by one or more substituents including, but notlimited to, alkyl, alkenyl, alkynyl, cycloalkyl, aroyl, halo, haloalkyl(e.g., trifluoromethyl), substituted or unsubstituted heterocycloalkyl,haloalkoxy (e.g., trifluoromethoxy), hydroxy, alkoxy, cycloalkyloxy,heterocylooxy, oxo, alkanoyl, aryl, substituted aryl, substituted orunsubstituted heteroaryl (e.g., indolyl, imidazolyl, furyl, thienyl,thiazolyl, pyrrolidyl, pyridyl, pyrimidyl and the like), arylalkyl,alkylaryl, heteroaryl, heteroarylalkyl, alkylheteroaryl, heterocyclo,aryloxy, alkanoyloxy, amino, alkylamino, arylamino, arylalkylamino,cycloalkylamino, heterocycloamino, mono- and di-substituted amino,alkanoylamino, aroylamino, aralkanoylamino, substituted alkanoylamino,substituted arylamino, substituted aralkanoylamino, carbamyl (e.g.,CONH₂), substituted carbamyl (e.g., CONH-alkyl, CONH-aryl,CONH-arylalkyl or instances where there are two substituents on thenitrogen), carbonyl, alkoxycarbonyl, carboxy, cyano, ester, ether,guanidino, nitro, sulfonyl, alkylsulfonyl, arylsulfonyl,arylalkylsulfonyl, sulfonamido (e.g. SO₂NH₂), substituted sulfonamido,thiol, alkylthio, arylthio, arylalkylthio, cycloalkylthio,heterocyclothio, alkylthiono, arylthiono and arylalkylthiono.

As used herein, and unless otherwise specified, the term “alkyl” means asaturated straight chain or branched non-cyclic hydrocarbon having 1-20carbon atoms, preferably 1-10 carbon atoms and most preferably 1-4carbon atoms. Representative saturated straight chain alkyls include-methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl,-n-octyl, -n-nonyl and -n-decyl; while saturated branched alkyls include-isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl,2-methylbutyl, 3-methylbutyl, 2-methylpentyl, 3-methylpentyl,4-methylpentyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl,5-methylhexyl, 2,3-dimethylbutyl, 2,3-dimethylpentyl,2,4-dimethylpentyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl,2,5-dimethylhexyl, 2,2-dimethylpentyl, 2,2-dimethylhexyl,3,3-dimtheylpentyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylpentyl,3-ethylpentyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl,2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, 2-methyl-4-ethylpentyl,2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2-methyl-4-ethylhexyl,2,2-diethylpentyl, 3,3-diethylhexyl, 2,2-diethylhexyl, 3,3-diethylhexyland the like. An alkyl group can be unsubstituted or substituted.Unsaturated alkyl groups include alkenyl groups and alkynyl groups,which are discussed below.

As used herein, and unless otherwise specified, the term “alkenyl” meansa straight chain or branched non-cyclic hydrocarbon having 2-20 carbonatoms, preferably 2-10 carbon atoms, most preferably 2-6 carbon atoms,and including at least one carbon-carbon double bond. Representativestraight chain and branched (C₂-C₁₀)alkenyls include -vinyl, -allyl,-1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl,-3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl,-1-hexenyl, -2-hexenyl, -3-hexenyl, -1-heptenyl, -2-heptenyl,-3-heptenyl, -1-octenyl, -2-octenyl, -3-octenyl, -1-nonenyl, -2-nonenyl,-3-nonenyl, -1-decenyl, -2-decenyl, -3-decenyl and the like. The doublebond of an alkenyl group can be unconjugated or conjugated to anotherunsaturated group. An alkenyl group can be unsubstituted or substituted.

As used herein, and unless otherwise specified, the term “alkynyl” meansa straight chain or branched non-cyclic hydrocarbon having 2-20 carbonatoms, preferably 2-10 carbon atoms, most preferably 2-6 carbon atoms,and including at lease one carbon-carbon triple bond. Representativestraight chain and branched (C₂-C₁₀)alkynyls include -acetylenyl,-propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl,-3-methyl-1-butynyl, -4-pentynyl, -1-hexynyl, -2-hexynyl, -5-hexynyl,-1-heptynyl, -2-heptynyl, -6-heptynyl, -1-octynyl, -2-octynyl,-7-octynyl, -1-nonynyl, -2-nonynyl, -8-nonynyl, -1-decynyl, -2-decynyl,-9-decynyl, and the like. The triple bond of an alkynyl group can beunconjugated or conjugated to another unsaturated group. An alkynylgroup can be unsubstituted or substituted.

As used herein, and unless otherwise specified, the term“pharmaceutically acceptable salt” refers to a salt prepared frompharmaceutically acceptable non-toxic acids or bases including inorganicacids and bases and organic acids and bases. Suitable pharmaceuticallyacceptable base addition salts for the compositions provided hereininclude, but are not limited to, metallic salts made from aluminum,calcium, lithium, magnesium, potassium, sodium, and zinc, or organicsalts made from lysine, N,N′-dibenzylethylenediamine, chloroprocaine,choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine)and procaine. Suitable non-toxic acids include, but are not limited to,inorganic and organic acids such as acetic, alginic, anthranilic,benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic,formic, fumaric, furoic, galacturonic, gluconic, glucuronic, glutamic,glycolic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic,mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic,phenylacetic, phosphoric, propionic, salicylic, stearic, succinic,sulfanilic, sulfuric, tartaric acid, and p-toluenesulfonic acid.Specific non-toxic acids include hydrochloric, hydrobromic, phosphoric,sulfuric, and methanesulfonic acids. Examples of specific salts thusinclude hydrochloride and mesylate salts. Others are well-known in theart, see e.g., Remington's Pharmaceutical Sciences, 18^(th) ed., MackPublishing, Easton Pa. (1990) or Remington: The Science and Practice ofPharmacy, 19th ed., Mack Publishing, Easton Pa. (1995).

As used herein, the term “hydrate” means a compound provided herein, ora salt thereof, that further includes a stoichiometric ornon-stoichiometric amount of water bound by non-covalent intermolecularforces.

As used herein, the term “clathrate” means a compound provided herein,or a salt thereof in the form of a crystal lattice that contains spaces(e.g., channels) that have a guest molecule (e.g., a solvent or water)trapped within.

As used herein, and unless otherwise indicated, the term “prodrug” meansa derivative of a compound that can hydrolyze, oxidize, or otherwisereact under biological conditions (in vitro or in vivo) to provide anactive compound. Examples of prodrugs include, but are not limited to,derivatives and metabolites of a compound that include biohydrolyzablemoieties such as biohydrolyzable amides, biohydrolyzable esters,biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzableureides, and biohydrolyzable phosphate analogues. Preferably, prodrugsof compounds with carboxyl functional groups are the lower alkyl estersof the carboxylic acid. Esterifying any of the carboxylic acid moietiespresent on the molecule conveniently forms the carboxylate esters.Prodrugs can typically be prepared using well-known methods, such asthose described by Burger's Medicinal Chemistry and Drug Discovery 6thed. (Donald J. Abraham ed., 2001, Wiley), and Design and Application ofProdrugs (H. Bundgaard ed., 1985, Harwood Academic Publishers Gmfh).

As used herein, and unless otherwise specified, the term “stable,” whenused in connection with a formulation, means that the active agent ofthe formulation, when prepared using the methods provided herein,remains solubilized for a specified amount of time and does notsignificantly degrade or aggregate or become otherwise modified (e.g.,as determined by HPLC).

As used herein, and unless otherwise specified, “temperature below theTc” is understood to be a temperature which is lower than the Tc of thelipid having the lowest Tc, and “temperature greater than the Tc” isunderstood to be a temperature which is greater than the Tc of the lipidhaving the highest Tc.

As used herein, and unless otherwise specified, the term “harmfulingredient,” when used in connection with pharmaceutical compositions,means an ingredient commonly used in a pharmaceutical composition thatmay cause clinical side effects such as, but not limited to, hemolysis,hypersensitive reaction, peripheral neuropathies, and/or decrease in thebioavailability of the active ingredient of the composition. Examples ofharmful ingredients include, but are not limited to: toxic solvents,including organic solvents such as ethanol, methanol, 1-propanol,2-propanol, acetone, acetonitrile, ethyl acetate, methyl acetate,diethyl ether, dimethyl ether, diisopropyl ether, methyl tert-butylether (“MTBE”), tetrahydrofuran (“THF”), dichloromethane, chloroform,carbon tetrachloride, 1,2-dicholroethane, pentane, hexanes, heptane,petroleum ether, dioxane, ethylene glycol, diethylene glycol, diglyme,1,2-dimethoxyethane, 1-butanol, 2-butanol, 2-butanone, benzene, toluene,dimethylsulfoxide (“DMSO”), dimethylformamide (“DMF”),hexamethylphosphoramide (“HMPA”), N-methylpyrrolidone, glycerin,nitromethane, triethyl amine, xylenes, CREMOPHOR® EL, and polyethyleneglycol (“PEG”); co-detergents or surfactants such as polysorbates (e.g.,Tweens) or vitamin E; oils such as Castor oil or corn oil; proteins suchas HSA; or any other biologic which is potential source ofcontamination.

DETAILED DESCRIPTION

Provided herein is a method for the preparation of liposomes, the methodcomprising:

(a) Combining one or more lipids in an aqueous medium at ambienttemperature;

(b) Dispersing the lipids in the aqueous medium; and

(c) Adding one or more sugars to the resulting mixture, thereby forminga solution of liposomes.

In another embodiment, at step (b), the method further compriseshomogenization of the lipids in the aqueous medium.

Some embodiments further comprise a step (d), wherein one or more activeagents is added to the solution of liposomes. In one embodiment, theactive agent is a hydrophobic drug. In one embodiment, the active agentis added as a solid. In another embodiment, the active agent is added inan organic solvent. In another embodiment, the active agent is added inorganic solvent which further comprises one or more fatty acid salts,fatty acids and/or phospholipids.

In another embodiment, the active agent is added during formation of theliposome.

In certain embodiments, the resulting liposomes are less than about 1 μmin diameter.

In one embodiment, the resulting liposomes are less than about 500 nm indiameter.

In one embodiment, the resulting liposomes are less than about 100 nm indiameter.

In one embodiment, at least one of the lipids is a phospholipid or amixture of phospholipids. Examples of phospholipids include, but are notlimited to, phosphatidic acid (“PA”), phosphatidylcholine (“PC”),phosphatidylglycerol (“PG”), phophatidylethanolamine (“PE”),phophatidylinositol (“PI”), and phosphatidylserine (“PS”), sphingomyelin(including brain sphingomyelin), lecithin, lysolecithin,lysophosphatidylethanolamine, cerebrosides,diarachidoylphosphatidylcholine (“DAPC”),didecanoyl-L-alpha-phosphatidylcholine (“DDPC”),dielaidoylphosphatidylcholine (“DEPC”), dilauroylphosphatidylcholine(“DLPC”), dilinoleoylphosphatidylcholine, dimyristoylphosphatidylcholine(“DMPC”), dioleoylphosphatidylcholine (“DOPC”),dipalmitoylphosphatidylcholine (“DPPC”), distearoylphosphatidylcholine(“DSPC”), 1-palmitoyl-2-oleoyl-phosphatidylcholine (“POPC”),diarachidoylphosphatidylglycerol (“DAPG”),didecanoyl-L-alpha-phosphatidylglycerol (“DDPG”),dielaidoylphosphatidylglycerol (“DEPG”), dilauroylphosphatidylglycerol(“DLPG”), dilinoleoylphosphatidylglycerol,dimyristoylphosphatidylglycerol (“DMPG”), dioleoylphosphatidylglycerol(“DOPG”), dipalmitoylphosphatidylglycerol (“DPPG”),distearoylphosphatidylglycerol (“DSPG”),1-palmitoyl-2-oleoyl-phosphatidylglycerol (“POPG”),diarachidoylphosphatidylethanolamine (“DAPE”),didecanoyl-L-alpha-phosphatidylethanolamine (“DDPE”),dielaidoylphosphatidylethanolamine (“DEPE”),dilauroylphosphatidylethanolamine (“DLPE”),dilinoleoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine(“DMPE”), dioleoylphosphatidylethanolamine (“DOPE”),dipalmitoylphosphatidylethanolamine (“DPPE”),distearoylphosphatidylethanolamine (“DSPE”),1-palmitoyl-2-oleoyl-phosphatidylethanolamine (“POPE”),diarachidoylphosphatidylinositol (“DAPI”),didecanoyl-L-alpha-phosphatidylinositol (“DDPI”),dielaidoylphosphatidylinositol (“DEPI”), dilauroylphosphatidylinositol(“DLPI”), dilinoleoylphosphatidylinositol,dimyristoylphosphatidylinositol (“DMPI”), dioleoylphosphatidylinositol(“DOPI”), dipalmitoylphosphatidylinositol (“DPPI”),distearoylphosphatidylinositol (“DSPI”),1-palmitoyl-2-oleoyl-phosphatidylinositol (“POPI”),diarachidoylphosphatidylserine (“DAPS”), didecanoyl-L-alpha-phosphatidylserine (“DDPS”),dielaidoylphosphatidylserine (“DEPS”), dilauroylphosphatidylserine(“DLPS”), dilinoleoylphosphatidylserine, dimyristoylphosphatidylserine(“DMPS”), dioleoylphosphatidylserine (“DOPS”),dipalmitoylphosphatidylserine (“DPPS”), distearoylphosphatidylserine(“DSPS”), 1-palmitoyl-2-oleoyl-phosphatidylserine (“POPS”), diarachidoylsphingomyelin, didecanoyl sphingomyelin, dielaidoyl sphingomyelin,dilauroyl sphingomyelin, dilinolcoyl sphingomyelin, dimyristoylsphingomyelin, sphingomyelin, dioleoyl sphingomyelin, dipalmitoylsphingomyelin, distearoyl sphingomyelin, and1-palmitoyl-2-oleoyl-sphingomyelin.

The phospholipids provided herein may be chiral or achiral. The chiralphospholipids provided herein may be D- or L-phospholipids, for example,L-α-phosphatidylcholine or L-3-phosphatidylcholine.

In one embodiment, L-α-phosphatidylcholine is used in the methodsprovided herein.

In another embodiment, provided herein is a method for the preparationof liposomes, the method comprising:

(a) combining sodium oleate and L-α-phosphatidylcholine in an aqueousmedium at ambient temperature;

(b) dispersing sodium oleate and L-α-phosphatidylcholine in the aqueousmedium; and

(c) adding one or more sugars to the resulting mixture, thereby forminga solution of liposomes.

In one embodiment, the aqueous medium contains one or more activeagents, or pharmaceutically acceptable salts, hydrates, clathrates orprodrugs thereof. Examples of active agents include, but are not limitedto, lapachone (β-lapachone), taxanes (including, but not limited to,taxol, 7-epitaxol, 7-acetyl taxol, 10-desacetyltaxol,10-desacetyl-7-epitaxol, 7-xylosyltaxol, 10-desacetyl-7-sylosyltaxol,7-glutaryltaxol, 7-N,N-dimethylglycycltaxol, 7-L-alanyltaxol, taxotere,and mixtures thereof), paclitaxel, colchicine, transferrin,cyclosporines, cyclosporin A, ketoprofen, propofol, acetylsalicylicacid, acetaminophen, amphotericin, digoxin, doxorubicin, daunorubicin,epirubicin, idarubicin, angiogenesis inhibitors (e.g, bevacizumab,ranibizumab, vitaxin, carboxyamidotriazole, combretastatin A-4,fumagillin analogs (e.g., TNP-470), CM101, IFN-α, interleukin-10,interleukin-12, platelet factor-4, suramin, SU5416, thrombospondin,VEGFR antagonists, angiostatin, endostatin, 2-methoxyestradiol,tecogalan, thalidomide, prolactin, linomide, angiopoietin-1, basicfibroblast growth factor, vascular endothelial growth factor),vinca-alkaloids (e.g., vinblastine, vincristine, vindesin, etoposide,etoposide phosphate, and teniposide), cytarabine, actinomycin,etoposide, bleomycin, gentamycin, cyclophosphamide, methotrexate,streptozotocin, cytosine, β-D-arabinofuranoside-5′-triphosphate,cytochrome C, cisplatin, N-phosphono-acetyl-L-aspartic acid,5-fluoroorotic acid, acyclovir, zidovudine, interferons,aminoglycosides, cephalosporins, tetracyclines, propranolol, timolol,labetolol, clonidine, hydralazine, imipramine, amitriptyline, doxepim,phenyloin, diphenhydramine, chlorphenirimine, promethazine,prostaglandins, methotrexate, progesterone, testosterone, estradiol,estrogen, epirubicin, beclomethasone and esters, vitamin E, cortisone,dexamethasone and esters, betamethasone valerete, biphenyl dimethyldicarboxylic acid, calcitonins, camptothecin, captopril, cephazoline,chloroquinine, chlorothiazole, co-agulation factors VIII and IX,d-alpha-tocopherol, dexamethasone, dichlofenac, etoposide, feldene,flubiprofen, 5-fluorouracil, fluoxetine, fusidic acid, gentamicin,glyburide, granisetron, growth hormones, indomethacin, insulin,itraconazole, ketoconazole, methotrexate, metronidazole, minoxidil,mitomycin, nafcillin, naproxen, ondansetron, oxyphenbutazone, parazosin,physostigmine, piroxicam, prednisolone, primaquine, quinine, ramipril,taxotane, tenoxicam, terazosin, triamcinolone, urokinase, opioidanalgesics (e.g., alfentanil, anileridine, codiene, diamorphine,fentanyl, hydrocodone, hydromorphone, meperidine, morphine, oxycodone,oxymorphone, propoxyphene, sufentanil, pentazocine and nalbuphine),non-steroidal anti-inflammatory drugs (e.g., aspirin, indometacin,ibuprofen, mefenamic acid and phenylbutazone), angiotensin convertingenzyme (“ACE”) inhibitors (e.g., captoprilpolyene), protein kinase Cinhibitors, antibiotics (e.g., imidazole and triazole antibiotics),folic acid, anthracycline antibiotics, anti-sense RNAs, tricathecums,microbial ribosomal-inactivating toxins (e.g., gelonin, abrin, ricin Achain, Pseudomonas exotoxin, diptheria toxin, pokeweed antiviralpeptide), pipecolic acid derivatives (e.g., tacrolimus), plantalkaloids, dyes, radioisotope-labeled compounds, radiopaque compounds,radiosensitizers (e.g., 5-chloro-2′-deoxyuridine,5-bromo-2′-deoxyuridine and 5-iodo-2′-deoxyuridine), fluorescentcompounds, mydriatic compounds, bronchodilators, local anesthetics(e.g., dibucaine and chlorpromazine), antifungal agents (e.g.,miconazole, terconazole, econazole, isoconazole, butaconazole,clotrimazole, itraconazole, nystatin, naftifine and amphotericin B),antiparasitic agents, hormones, hormone antagonists, immunomodulators,neurotransmitter antagonists, anti-diabetic agents, antiglaucoma agents,vitamins, narcotics, and imaging agents. For additional disclosure ofactive agents, see Gilman et al., Goodman and Gilman's: ThePharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York,2001; The Merck Manual of Diagnosis and Therapy, Berkow, M. D. et al.(eds.), 17th Ed., Merck Sharp & Dohme Research Laboratories, Rahway,N.J., 1999; Cecil Textbook of Medicine, 20th Ed., Bennett and Plum(eds.), W.B. Saunders, Philadelphia, 1996.

In one embodiment, the active agent is a hydrophobic compound, or acompound with poor solubility in water.

In another embodiment, the active agent is a non-hydrophobic compound.

In one embodiment, the active agent is a water solublemembrane-impermeant agent such as a peptide, a protein, a nucleic acid,a nucleotide, a nucleoside, a carbohydrate or an analog thereof.

In one embodiment, the aqueous medium does not contain an active agent.

In another embodiment, the resulting solution contains 10% by weighttrehalose.

In another embodiment, the active agent is lapachone, or apharmaceutically acceptable salt, hydrate, clathrate or prodrug thereof.

In another embodiment, the active agent is transferrin, or apharmaceutically acceptable salt, hydrate, clathrate or prodrug thereof.

In another embodiment, the active agent is cyclosporine, or apharmaceutically acceptable salt, hydrate, clathrate or prodrug thereof.

In one embodiment, the active agents are transferrin and lapachone, orpharmaceutically acceptable salts, hydrates, clathrates or prodrugsthereof.

Examples of sugars that may be used in the methods provided hereininclude, but are not limited to, sucrose, glucose, fructose, lactose,maltose, mannose, galactose and trehalose.

In one embodiment, the sugar is trehalose.

In one embodiment, the liposomal preparation is suitable for parenteraladministration to a patient suffering from one or more diseases ordisorders.

In one embodiment, the patient is a human.

In certain embodiments, the sequence of the addition of active agent(s)results in enhanced solubility of the active agent(s). The conventionalmethod of incorporating hydrophobic drug to liposome is by adding thedrug to lipid before liposome preparation. See, e.g., Immordino, M. L.et al., Journal of Controlled Release, 2003, 91: 417-429. By theconventional process, the incorporation of the drug is only 0.3 to 0.7mg/mL.

Provided herein are methods for the preparation of liposomes wherein theaddition of the active agent(s) after fog illation of the liposomeresults in enhanced solubility of the active agent(s). In oneembodiment, the solubility of the active agent(s) in liposome isincreased by at least about two-fold, five-fold or ten-fold compared tothe conventional process. In one embodiment, the solubility of theactive agent(s) in liposome is increased to about 5 mg/mL.

In certain embodiments in which the active agent is a hydrophobic drug,the active agent is added to pre-made liposome as a solid or in anorganic solvent. In one embodiment, the pre-made liposome comprises oneor more fatty acid salts, fatty acids and/or phospholipids to increasethe solubility of the active agent.

In one embodiment, the sequence of the addition of active agent(s)results in greater efficiency of incorporation of the active agent(s)into liposome. In certain embodiments, the efficiency of incorporationinto liposome is 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100%. Incertain embodiments, the efficiency of incorporation is 90%, 95%, 98%,99% or 100%.

Without being limited to a particular theory or mechanism, the increasein incorporation of active agent to pre-made liposome (e.g., to aconcentration of about 5 mg/mL) may be due to increase in surface areaof liposome.

Also provided herein is a liposomal composition prepared by a methodcomprising:

(a) Combining one or more lipids in an aqueous medium at ambienttemperature;

(b) Dispersing the lipids in the aqueous medium; and

(c) Adding one or more sugars to the resulting mixture, thereby forminga solution of liposomes.

In another embodiment, provided herein is a liposomal compositionprepared by a method comprising:

(a) Combining one or more lipids in an aqueous medium at ambienttemperature;

(b) Dispersing the lipids in the aqueous medium;

(c) Adding one or more sugars to the resulting mixture, thereby forminga solution of liposomes; and

(d) Adding an active agent to the solution of liposomes.

In one embodiment, the active agent is a hydrophobic drug.

In one embodiment, the active agent is added as a solid.

In one embodiment, the active agent is added in an organic solvent.

In one embodiment, the active agent in organic solvent further comprisesone or more fatty acid salts, fatty acids and phospholipids.

In certain embodiments, the methods provided herein result in stablesolutions, compositions or formulations comprising liposomes and one ormore active agents. In these embodiments, the active agent(s) remainsolubilized for a specified amount of time and do not significantlydegrade, aggregate or become otherwise modified (e.g., as determined byHPLC).

In some embodiments, about 70 percent or greater, about 80 percent orgreater or about 90 percent or greater of the active agent remainssolubilized after a week after dilution with an acceptable diluent at anelevated temperature (e.g., about 35° C. or higher).

In other embodiments, about 70 percent or greater, about 80 percent orgreater or about 90 percent or greater of the active agent remainssolubilized after a week after dilution with an acceptable diluent atroom temperature.

In other embodiments, about 70 percent or greater, about 80 percent orgreater or about 90 percent or greater of the active agent remainssolubilized after a week at a reduced temperature (e.g., about 10° C. orlower).

In certain embodiments, the methods provided herein result in enhancedsolubility of an active agent, as compared to the solubility of the sameactive agent in an aqueous medium. Specifically, when the methodsprovided herein are used, the solubility of the active agent increasesabout 20 percent or more, about 40 percent or more, about 60 percent ormore, about 80 percent or more, about 100 percent or more, or about 200percent or more of the solubility of the same active agent in areference solvent. In some embodiments, the reference solvent is water.

Also provided herein is a method of treating a disease or disorder usinga liposomal composition provided herein. In some embodiments, thedisease or disorder includes, but is not limited to, oncologicaldisorders, proliferative disorders, central nervous system disorders,autoimmune disorders, and inflammatory diseases or disorders. In otherembodiments the methods are directed to the treatment of bacterial,viral or fungal infections.

Proliferative disorders (e.g. cancer) that may be treated by the methodsprovided herein include, but are not limited to, neoplasms, tumors(malignant and benign) and metastases, or any disease or disordercharacterized by uncontrolled cell growth. The cancer may be a primaryor metastatic cancer. Specific examples of cancers that can beprevented, managed, treated or ameliorated in accordance with themethods of the invention include, but are not limited to, cancer of thehead, neck, eye, mouth, throat, esophagus, chest, bone, lung, colon,rectum, stomach, prostate, breast, ovaries, kidney, liver, pancreas, andbrain. Additional cancers include, but are not limited to, thefollowing: leukemias (e.g., acute leukemia, acute lymphocytic leukemia),acute myelocytic leukemias (e.g., myeloblastic, promyelocytic,myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplasticsyndrome), chronic leukemias (e.g., chronic myelocytic (granulocytic)leukemia, chronic lymphocytic leukemia, hairy cell leukemia),polycythemia vera, lymphomas (e.g., Hodgkin's disease, non-Hodgkin'sdisease), multiple myelomas (e.g., smoldering multiple myeloma,nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia,solitary plasmacytoma and extramedullary plasmacytoma), Waldenstrom'smacroglobulinemia, monoclonal gammopathy of undetermined significance,benign monoclonal gammopathy, heavy chain disease, bone and connectivetissue sarcomas (e.g., bone sarcoma, osteosarcoma, chondrosarcoma,Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone,chordoma, periosteal sarcoma, soft-tissue sarcomas, angiosarcoma(hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma,liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovialsarcoma), brain tumors (e.g., glioma, astrocytoma, brain stem glioma,ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma,craniopharyngioma, medulloblastoma, meningioma, pineocytoma,pineoblastoma, primary brain lymphoma), breast cancer (e.g.,adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma,medullary breast cancer, mucinous breast cancer, tubular breast cancer,papillary breast cancer, Paget's disease, and inflammatory breastcancer), adrenal cancer (e.g., pheochromocytom and adrenocorticalcarcinoma), thyroid cancer (e.g., papillary or follicular thyroidcancer, medullary thyroid cancer and anaplastic thyroid cancer),pancreatic cancer (e.g., insulinoma, gastrinoma, glucagonoma, vipoma,somatostatin-secreting tumor, and carcinoid or islet cell tumor),pituitary cancers (e.g., Cushing's disease, prolactin-secreting tumor,acromegaly, and diabetes insipius), eye cancers (e.g., ocular melanomasuch as iris melanoma, choroidal melanoma, and cilliary body melanoma,and retinoblastoma), vaginal cancers (e.g., squamous cell carcinoma,adenocarcinoma, and melanoma), vulvar cancer (e.g., squamous cellcarcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, andPaget's disease), cervical cancers (e.g., squamous cell carcinoma, andadenocarcinoma), uterine cancers (e.g., endometrial carcinoma anduterine sarcoma), ovarian cancers (e.g., ovarian epithelial carcinoma,borderline tumor, germ cell tumor, and stromal tumor), esophagealcancers (e.g., squamous cancer, adenocarcinoma, adenoid cycticcarcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma,melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell)carcinoma), stomach cancers (e.g., adenocarcinoma, fungaling (polypoid),ulcerating, superficial spreading, diffusely spreading, malignantlymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma), colon cancers,rectal cancers, liver cancers (e.g., hepatocellular carcinoma andhepatoblastoma, gallbladder cancers such as adenocarcinoma),cholangiocarcinomas (e.g., pappillary, nodular, and diffuse), lungcancers (e.g., non-small cell lung cancer, squamous cell carcinoma(epidermoid carcinoma), adenocarcinoma, large-cell carcinoma andsmall-cell lung cancer), testicular cancers (e.g., germinal tumor,seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma,embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sactumor), prostate cancers such as but not limited to, adenocarcinoma,leiomyosarcoma, and rhabdomyosarcoma), penile cancers, oral cancers(e.g., squamous cell carcinoma), basal cancers, salivary gland cancers(e.g., adenocarcinoma, mucoepidermoid carcinoma, and adenoidcysticcarcinoma), pharynx cancers (e.g., squamous cell cancer, and verrucous),skin cancers (e.g., basal cell carcinoma, squamous cell carcinoma andmelanoma, superficial spreading melanoma, nodular melanoma, lentigomalignant melanoma, acral lentiginous melanoma), kidney cancers (e.g.,renal cell cancer, adenocarcinoma, hypernephroma, fibrosarcoma,transitional cell cancer (renal pelvis and/or uterer)), Wilms' tumor,bladder cancers (e.g., transitional cell carcinoma, squamous cellcancer, adenocarcinoma, carcinosarcoma), myxosarcoma, osteogenicsarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma,synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma,bronchogenic carcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma and papillary adenocarcinomas, follicularlymphomas, carcinomas with p53 mutations, hormone dependent tumors ofthe breast, prostate and ovary, precancerous lesions such as familialadenomatous polyposis, and myelodysplastic syndromes.

Other specific diseases and disorders that may be treated by the methodsprovided herein include, but are not limited to, the following: allergicdisorders, inflammation, asthma, arthritis, encephalitis, rheumatoidarthritis, osteoarthritis, psoriatic arthritis, inflammatory osteolysis,chronic or acute obstructive pulmonary disease, chronic or acutepulmonary inflammatory disease, inflammatory bowel disease, Crohn'sDisease, gout, Bechet's Disease, Henoch-Schonlein purpura (“HSP”),septic shock, sepsis, meningitis, colitis, inflammation due toreperfusion, psoriasis, fibrosis including pulmonary fibrosis,Parkinson's disease, bradykinesia, muscle rigidity, Parkinsonian tremor,Parkinsonian gait, motion freezing, depression; defective long-termmemory, Rubinstein-Taybi syndrome (RTS), dementia, sleep disorders,insomnia, postural instability, hypokinetic disorders, hyperkineticdisorders, synuclein disorders, multiple system atrophies, striatonigraldegeneration, olivopontocerebellar atrophy, Shy-Drager syndrome, motorneuron disease with parkinsonian features, Lewy body dementia, Taupathology disorders, progressive supraneulear palsy, corticobasaldegeneration, frontotemporal dementia; amyloid pathology disorders, mildcognitive impairment, Alzheimer disease, Alzheimer disease withparkinsonism, Wilson disease, Hallervorden-Spatz disease,Chediak-Hagashi disease, SCA-3 spinocerebellar ataxia, X-linked dystoniaparkinsonism, Huntington disease, prion disease, chorea, ballismus,dystonia tremors, Amyotrophic Lateral Sclerosis (“ALS”), CNS trauma,myoclonus, and diseases or disorders associated with undesired immunereaction (e.g., organ rejection associated with an organ transplant).

Viral infections that may be treated by the methods provided hereininclude, but are not limited to, the following: human immunodeficiencyvirus (“HIV”), herpes simplex virus type 1, herpes simplex virus type 2,influenza viruses, influenza virus type A, influenze virus type B,parainfluenza virus, human papillomavirus (“HPV”), adenoviruses,rhinoviruses, hepatitis A virus, hepatitis B virus, hepatitis C virus,hepatitis D virus, hepatitis E virus, dengue fever, yellow fever, WestNile virus, Japanese encephalitis virus, GB virus A, GB virus-B, GBvirus-C, bovine viral diarrhea virus (“BVDV”), classical swine fevervirus (i.e., hog cholera virus), border disease virus, varicella zostervirus, smallpox, measles, rabies virus, arbovirus, cytomegalovirus,mumps virus, poliovirus, coxsackie B virus, Epstein-Barr virus, rubellavirus, parvovirus B19, coronaviruses (e.g., SARS coronavirus),astrovirus, norovirus, rotavirus, and adenoviruses.

Fungal infections that may be treated by the methods provided hereininclude, but are not limited to, aspergillosis, blastomycosis,coccidioidomycosis, cryptococcosis, fungal sinusitis, histoplasmosis,hypersensitivity pneumonitis, mucormycosis, paracoccidioidomycosis,sporotrichosis, and Valley Fever.

Bacterial infections that may be treated by the methods provided hereininclude, but are not limited to, brucellosis, cholera, leprocy,leptospirosis, shigellosis, trench fever, tularemia, Q fever, Whitmore'sdisease, yersiniosis, yaws, vibrio vulnificus infections, streptococcusinfections, staphylococcus infections and E. coli infections.

EXAMPLES Example 1 Preparation of Liposomes with No Drug: 6%L-α-Phosphatidylcholine (Soy) Liposome

6 g of L-α-phosphatidylcholine (Soy) was dispersed in 100 mL of waterusing a magnetic stirrer at 200 rpm for 10 minutes at ambienttemperature. The dispersed liposome (multilayer) was passed through aMicrotluidic homogenizer at 15,000 psi. Three cycles of passing resultedin a liposome less than 100 nm in diameter. Trehalose was then added tothe liposome to a final concentration of 10% (w/w). The resulting stableisotonic liposome was either used as liquid or lyophilized.

Example 2 Preparation of Liposomes Encapsulated with Lapachone

200 mg of lapachone and 6 g of L-α-phosphatidylcholine (Soy) weredispersed in 100 mL of water using a magnetic stirrer at 200 rpm for 10minutes at ambient temperature. The dispersed liposome (multilayer) waspassed through a Microfluidic homogenizer at 15,000 psi. Three cycles ofpassing resulted in a liposome encapsulated with 2 mg/mL lapachone lessthan 100 nm in diameter. Trehalose was then added to the liposome to afinal concentration of 10% (w/w). The resulting stable isotonic liposomeencapsulated with lapachone was either used as liquid or lyophilized.

Example 3 Preparation of Liposomes Encapsulated and Micro Emulsifiedwith Cyclosporine

500 mg of cyclosporine in 5 mL Mygliol and 6 g ofL-α-phosphatidylcholine (Soy) were dispersed in 100 mL of water using amagnetic stirrer at 200 rpm for 10 minutes at ambient temperature. Thedispersed liposome (multilayer) was passed through Microfluidichomogenizer at 15,000 psi. Three cycles of passing resulted in aliposome encapsulated with 5 mg/mL cyclosporine less than 100 nm indiameter. Trehalose was then added to the liposome to a finalconcentration of 10% (w/w). The resulting stable isotonic liposomeformulation encapsulated and micro emulsified with cyclosporine waseither used as liquid or lyophilized.

Example 4 Preparation of Liposomes Encapsulated with Transferrin

200 mg of transferrin and 6 g of L-α-phosphatidylcholine (Soy) weredispersed in 100 mL of water using a magnetic stirrer at 200 rpm for 10minutes at ambient temperature. The dispersed liposome (multilayer) waspassed through a Microfluidic homogenizer at 15,000 psi. Three cycles ofpassing resulted in a liposome encapsulated with 5 mg/mL transferrinless than 100 nm in diameter. Trehalose was then added to the liposometo a final concentration of 10% (w/w). The resulting stable isotonicliposome formulation encapsulated with transferrin was either used asliquid or lyophilized.

Example 5 Preparation of Liposomes Encapsulated with Colchicine

6 mg of sodium oleate, 10 g of trehalose and 6 g L-α-phosphatidylcholine(Soy) were dispersed in 100 mL of water using a magnetic stirrer at 200rpm for 10 minutes at ambient temperature. The dispersed liposome(multilayer) was passed 10 times through a Microfluidic homogenizer at15,000 psi. 100 μL of colchicine dissolved in acetone was spiked into 1mL of the pre-made liposome and lyophilized. The resulting stableisotonic lyophilized liposome encapsulated with the drug is essentiallyfree from organic solvent after lyophilization. After lyophilization,the product can be reconstituted as a 1 mg/mL, 2 mg/mL, 3 mg/mL or 4mg/mL aqueous solution (e.g., Water For Injection).

All references cited herein are incorporated herein by reference intheir entireties and for all purposes to the same extent as if eachindividual publication, patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

What is claimed is:
 1. A method of preparing a pharmaceuticalcomposition comprising unilamellar liposomes, wherein the unilamellarliposomes comprise a hydrophobic pharmaceutical agent, the methodcomprising: (a) forming a suspension of multilamellar liposomes bydispersing one or more solid phospholipids in an aqueous medium, whereinthe aqueous medium is free of organic solvents and protein excipients,and the aqueous medium is at ambient temperature; (b) adding adisaccharide to the multilamellar liposome suspension, rendering thesuspension isotonic; (c) homogenizing the multilamellar liposomesuspension of step (b) to form an isotonic suspension of unilamellarliposomes; (d) adding the hydrophobic pharmaceutical agent as a solid tothe isotonic suspension of unilamellar liposomes and subjecting themixture to microfluidization to form the pharmaceutical compositioncomprising unilamellar liposomes, wherein the unilamellar liposomes areless than about 100 nm in diameter and comprise the hydrophobicpharmaceutical agent.
 2. The method of claim 1, wherein the homogenizingis carried out with a microfluidic homogenizer.
 3. The method of claim1, wherein the one or more phospholipids compriseL-α-phosphatidylcholine.
 4. The method of claim 1, wherein thedisaccharide is trehalose.
 5. The method of claim 4, wherein thetrehalose concentration of the pharmaceutical composition is 10% byweight.
 6. The method of claim 1, wherein the hydrophobic pharmaceuticalagent is lapachone, colchicine, cyclosporine, transferrin, or apharmaceutically acceptable salt thereof.
 7. The method of claim 1,wherein the hydrophobic pharmaceutical agent is lapachone, or apharmaceutically acceptable salt thereof.
 8. The method of claim 1,wherein the hydrophobic pharmaceutical agent is colchicine, or apharmaceutically acceptable salt thereof.
 9. The method of claim 1,wherein the hydrophobic pharmaceutical agent is cyclosporine, or apharmaceutically acceptable salt thereof.
 10. The method of claim 1,wherein each of steps (a)-(d) is performed without the application ofheat.
 11. The method of claim 1, wherein the method is performed withoutthe use of inorganic salts.
 12. The method of claim 1, wherein the oneor more solid phospholipids is soy phosphatidylcholine.
 13. A method ofpreparing a pharmaceutical composition comprising unilamellar liposomes,wherein the liposomes comprise a hydrophobic pharmaceutical agent, themethod comprising: (a) forming liposomes by concurrently mixing one ormore solid phospholipids, a disaccharide, and the hydrophobicpharmaceutical agent added as a separate component in solid form in anaqueous medium to produce multilamellar liposomes comprising thehydrophobic pharmaceutical agent, wherein the aqueous medium is free oforganic solvents, surfactants, and protein excipients, and the aqueousmedium is at ambient temperature; and (b) homogenizing the compositionof (a) to form the pharmaceutical composition comprising unilamellarliposomes, wherein the unilamellar liposomes are less than about 100 nmin diameter and comprise the hydrophobic pharmaceutical agent.
 14. Themethod of claim 1, wherein the phospholipid is hydrogenatedL-α-phosphatidylcholine.
 15. The method of claim 13, wherein step (b) isperformed without applying heat.
 16. The method of claim 1, wherein theconcentration of the hydrophobic pharmaceutical agent in the unilamellarliposomes is at least about 3.5 mg/mL.
 17. The method of claim 1,wherein the concentration of the hydrophobic pharmaceutical agent in theunilamellar liposomes is about 5 mg/mL.
 18. The method of claim 1,further comprising (e) homogenizing the pharmaceutical composition. 19.The method of claim 18, further comprising (f) lyophilizing thepharmaceutical composition.
 20. The method of claim 1, wherein addingthe hydrophobic active agent comprises adding more than one active agentto the dispersed unilamellar liposomes.
 21. The method of claim 13,wherein the hydrophobic pharmaceutical agent is lapachone, colchicine,cyclosporin, transferrin, or a pharmaceutically acceptable salt thereof.